Corticosteroid binding globulin, hereinafter referred to as "CBG", is a plasma glycoprotein having a high affinity for endogenous glucocorticoids. CBG functions to regulate the biological activity of glucocorticoids in vivo. The binding of glucocorticoids to CBG is believed to reduce the metabolic clearance rate of glucocorticoids from plasma, thereby increasing their biological half-life. Many glucocorticoids such as steroid hormones exhibit anti-inflammatory properties. Upon binding with CBG, glucocorticoids become biologically inactivated and, thus, do not exert anti-inflammatory action. However, at sites of inflammation where glucocorticoid activity is required, glucocorticoids are enzymatically released from their CBG-bound form to assume their free active form in which they function in the capacity of an anti-inflammatory agent.
As a result of its high affinity for steroid hormones and its function in the transport of bound steroids to sites of inflammation, CBG has been found to be useful in the therapeutic treatment of inflammation by serving as a vehicle for the delivery of anti-inflammatory agents to sites of inflammation in mammals. This use has been described in U.S. Pat. Nos. 4,997,814 and 5,086,039, both of which are incorporated herein by reference.
CBG is subject to N-linked glycosylation, a common post-translational modification of proteins in eucaryotic cells. Native human CBG is highly glycosylated and carbohydrates have been reported to comprise approximately 25% of the total molecular weight of CBG (Akhrem et al., Biochim. Biophys. Acta; 1982, 714:177). Such extensive glycosylation leads to considerable size heterogeneity in native CBG. The heterogeneity of CBG results not only from the variation apt to occur in its glycosylation pattern but also the variability in the oligosaccharides binding to the various sites of glycosylation. Size heterogeneity is a feature that is undesirable with regard to the approval of CBG as a pharmaceutical because CBG is not presented as a conclusively defined product. Moreover, a composition comprising CBG may be deemed to be impure due to the variability in the constitution of CBG.
Deglycosylation of CBG appears to be an inappropriate route by which to render a fully defined CBG product as the biological activity of CBG is regulated, at least in part, by its glycosylated sites. Glycosylation is believed to regulate blood levels of CBG (Hossner et al., Endocrinology, 1981, 108:1780). Glycosylation also appears to be essential in retaining the conformation of active CBG i.e. the tertiary structure of CBG (Ghose-Dastidar et al., Proc. Natl. Acad. Sci. USA, 1991, 88:6408). Further, glycosylation appears to contribute to the molecular and cellular recognition of CBG (Rosner, Endocrine Rev., 1990, 11:65, Strel'chyonok et al., J. Steroid Biochem. Molec. Biol. 1991, 40:795). Although deglycosylation of CBG has been reported not to affect the biological activity of CBG (Mickelson et al. Biochemistry, 1982, 21:644), it was acknowledged at the time that the exoglycosidases utilized in the deglycosylation did not completely remove the carbohydrates attached at all sites of glycosylation.
In accordance with the standards required to obtain approval of CBG as a pharmaceutical, it would be desirable to eliminate components of native glycosylated CBG which are superfluous to its use in the treatment of inflammation. In so doing, a more appropriate CBG product may be provided for pharmaceutical application which is homogeneous in nature and the constitution of which is more thoroughly defined.